There is evidence that FKBP12.6 binds tightly to the cardiac ryanodine receptor (RyR2) and thereby modulates the release of Ca2+ from the sarcoplasmic reticulum (SR) during excitation-contraction coupling (2). It has been suggested that dissociation of FKBP12.6 from RyR2 occurs in heart failure and leads to elevated diastolic Ca2+ leak, reduced SR Ca2+ content, contractile dysfunction and arrhythmia generation (1). Our single-channel studies reveal that RyR2 activity may be controlled by the opposing actions of FKBP12.6 and FKBP12. To investigate how these proteins regulate RyR2 function in cardiac cells we have examined how removing FKBP12 and FKBP12.6 and then replacing both proteins affects waves of Ca2+-induced Ca2+-release (CICR) in isolated, permeabilised rat ventricular myocytes. FKBP12 and 12.6 were cloned, recombinantly expressed in Escherichia coli and purified. Wistar rats were killed by cervical dislocation, the cardiomyocytes were isolated and permeabilised by incubation with β-escin (0.1 mg/ml (3)) for 1 min. The permeabilised cells were resuspended in the mock intracellular solution with the following composition (mM): 100 KCl, 5 Na2ATP, 5.4 MgCl2, 25 Hepes, 0.1 K2EGTA, pH 7.0. Fluorescence from Fluo 5F (10 µM) was monitored by confocal microscopy using the BioRad MRC 1024 in linescan mode. Spontaneous waves of CICR were induced by 234 nM Ca2+. Unless otherwise stated, Student’s t-test was used to assess the difference between mean values. After treatment with 20 µM rapamycin (to remove FK binding proteins) for 4-6 minutes the frequency of the waves decreased (from 0.59±0.05 Hz to 0.13±0.04 Hz; S.E.M; n=8; P<0.001). Rapamycin treatment also led to the appearance of “mini-waves” that did not propagate properly throughout the cell and the baseline Fluo-5F fluorescence intensities increased to 204±47% (S.E.M.; n=8; P<0.05) of control. Adding back a physiological level of FKBP12 (3 µM) to the cell perfusate significantly increased wave frequency (from 0.13±0.04 Hz to 0.27±0.05 Hz; S.E.M; n=8; P<0.05) but did not fully reverse the effects of rapamycin. In contrast, FKBP12.6 (200 nM) had no significant effect on wave frequency (from 0.13±0.04 Hz (n=8) to 0.16±0.05 Hz (n=4) S.E.M; P<0.65). FKBP12 also fully reversed the rapamycin-induced increase in baseline fluorescence (n=8; P<0.05, Mann-Whitney Rank Sum test). Our results show that rapamycin treatment disrupts Ca2+-wave propagation suggesting that FK-binding proteins are essential for cardiac CICR. Adding back FKBP12.6 or FKBP12 causes more fully-propagating waves but only the addition of FKBP12 causes an increase in the frequency of Ca2+-waves. This may be related to the ability of FKBP12 to increase RyR2 open probability.